Luminescent coumarins have found widespread use as photosensitizers, laser dyes or pH indicators in biochemistry and medicine. There is therefore a huge amount of experimental and theoretic data about the luminescence characteristics, photophysics and photochemistry of coumarin derivatives. As for the similar application of the chemically strongly related 2-quinolones (carbostyrils), which can be considered as aza analogues of the coumarins and also luminescent, there is comparatively much less application data in literature. Though it is just the carbostyrils who should be more photo-stable and chemically more inert than the coumarin derivatives.
This relative lack of applications as luminescence markers is partly to be attributed to the fact that, much unlike the coumarins, despite hundreds of well-known carbostyril derivatives, hitherto obviously nobody has succeeded in shifting the absorption wavelength, compared to the unsubstituted form (330 nm), much beyond the value of 350 nm in the longer wave area and at the same time in obtaining a strong increase of the luminescence quantum yield. In case of coumarin, which in the unsubstituted state absorbs at shorter wavelengths than carbostyril, this succeeds easily by introducing electron delivering amino or ether substituents, mainly in position 7.
In contrast to this, for example, in the series of the 4-methylcarbostyrils the long wave UV absorption maximum of 331 nm measured in dimethylsulfoxide is shifted by an additional methoxy function in position 7 even to a slightly shorter wavelength (328 nm).
The production of photo-stable and strongly luminescent carbostyrils with absorption maxima over 350 nm would be of essential interest because of the more efficient elimination of disturbing, short-wave-absorbing foreign fluorophores in a complex matrix. Important applications in measuring technology will result for long wave absorbing carbostyrils thanks to extremely inexpensive LEDs, which have been recently available commercially, and which in the near UV area emit at around 370 nm and therefore in the near future will be important especially in the sensor development.
The interest in luminescent dyes has recently concentrated on analytic applications in biochemistry. Among the most promising applications there is the use of such chromophores for the production of lanthanide chelates, above all such with europium and terbium ions. To the chromophores used belong among others already determined carbostyrils, especially N-acyl derivatives of the 7-amino-4-methyl-2(1H)-quinolone (carbostyril 124). This has been described by M. Li and P. R. Selvin for example in J. Am. Chem. Soc., 117 (1995) 8132 and Bioconjugate Chem., 8 (1997) 127. The excitation in this case happens at 337 nm. The possibility of time-resolved measurements of the long-wave lanthanide emission makes such complexes attractive, particularly in biologic systems. They can for example be applied as favourable alternatives to the radioactive markings (radioimmunoassays, RIA) and simple fluoroimmunoassays (FIA). The so-called DELFIA(copyright) test (dissociation enhanced lanthanide fluorescence immunoassay) already represents a routine method. A further application of the time-resolved measurement of lanthanide complexes is the use of these chelates as luminescence markers by covalent binding with analytes in biological matrix.
The present invention resolves the problem of providing longer wave absorbing and strongly luminescent carbostyrils with UV maxima above 350 nm by the introduction of a very specific specimen of substituents with the general formula I. They are 4-trifluoromethyl substituted quinoline-2-ones with essentially two substituents or functionalities in position 6 and 7 bound via oxygen or nitrogen, which together cause long-wave absorption maxima above 350 nm and high emission quantum yields in combination with sufficient Stokes shifts. This could not be foreseen for the long-wave maximum because of the in itself rather disadvantageous effect of a methoxy group alone in position 7 (as described above).
In such a structure 1, without significant change of the luminescence properties, for example in position 1 and 3 additional substituents may be present that are suitable for the introduction of various other functionalities. Equally, in position 6 and 7 the residues R1 and R2 may have useful functions, suitable for complexing a metal ion or for the binding with reaction-capable analytes or for the immobilization on solid materials.
The potential of compounds of formula I is explained according to the invention by the bond of various side chains and functionalities and the measurement of the absorption properties as well as of the luminescence characteristic. The side chains for example allow the complex-like binding of europium (III) ions as well as the immobilization of these complexes on an analyte or at a solid matrix. For example, the nitration of the 4-trifluoro-methyl-6,7-dimethoxycarbostyril leads to the 3-nitro derivative that after reduction gives the corresponding 3-amino derivate. An N(1)-methylation of the nitro compound and following reduction to the analogous N(1)-methyl-3-amino compound or N(1)-benzyl-3-amino compound or the N(1)-phthalimidomethyl compound hardly changes the spectral properties. Therefore, also position 1 is suitable for bonding further functions, which for example can lead to immobilization later. For example already the 3-amino-4-trifluoromethyl-6,7-dimethoxycarbostyril can be acylated with a suitable anhydride (e.g. diethylenetriaminepentaacetate dianhydride xe2x80x9cDTPAxe2x80x9d) in position 3 on nitrogen. After hydrolysis a tetracarboxylic acid arises, which can be easily complexed with europium ions. Alternatively, before the hydrolysis of the anhydride, one can apply a linker either directly with a suitable nucleophile or via a second intermediate, that for its part can bind the finished complex to an analyte or to a further chromophore or to a fixed matrix.
For example, the mono anhydride formed with DTPA and carbostyril can be further activated by prolonged heating and hence additional formation of a 6-membered cyclic imide with the former 3-amino nitrogen of the trifluoromethyl-carbostyril (formula IIb in example 6). Easy selective hydrolysis of the anhydride function in IIb yields quantitatively an imine IId, which is capable to react with nucleophiles such as amines and hydrazines to form compounds of type IIIa. Hence selective activation and subsequent reaction of different carboxylic acid functions via the key DTPA intermediate IIa is possible.
The complexing with lanthanide ions can take place according to the demand before or after the completion of the total molecule. According to structure 1, the explanations above as well as the examples quoted later, it is obvious that complex-forming side chains at one of the points of the molecule indicated by R lead to compounds that still show the characteristic according to the invention of a desired UV absorption beyond 350 nm.
The substantial photo-physical properties of the example compound (III) complexed with Eu3+ and of the underlying chromophore (1) are reproduced in FIG. 1 and in the experimental part of the examples. From this it is clearly visible that after excitation at 370 nm of the europium complexes, besides the strong inherent fluorescence of the carbostyril, a marked transfer of the excitation to the complexed europium ion takes place (so-called antenna effect). Such a transfer of the excitation energy to europium has hardly been observed so far in this high absorption wavelength range, that is, after irradiation with light with wavelengths above 370 nm. The range considered efficient so far was between around 300 and 350 nm. Europium ions themselves are luminescent alone, that is, without transferring chromophore, only extremely weakly.
From the examples it is also clear, that an additional covalently bound or only added dye with a different absorption wavelength(e. g. 550 nm) does not disturb this process. In contrast to this, a dye, which absorbs in the range of the europium emission (570-710 nm), acts in a dynamic quenching way (luminescence intensity and decay time diminish) and therefore makes possible the construction of almost any sensor. The only condition that has to be fulfilled is that such an additional dye acts as an indicator for the analyte or parameter to be measured, such as the hydrogen ion concentration. Similar effects result naturally also from the protonation/deprotonation of the carbostyril chromophore on the carbonyl function or on a nitrogen atom bound directly to the carbostyril, because with this its spectral properties and consequently its antenna effect are changed. Time-resolved measurement of the emission spectrum after around a microsecond exclusively shows europium bands (without short-lived basic fluorescence), whose drop in a time lapse of around 2 milliseconds can also become the measuring principle. This proves the applicability of this complex type for time-resolved luminescence measurements also on a strongly fluorescence background.
Some of the new europium(III) complexes of 4-trifluoromethylcarbostyril derivatives are pH-sensitive. For example the complex of IIIb is an indicator in the range between pH 7 and 9 with a pKa of approximately 8.2. In the range between pH 7 and pH 4 there is a flat maximum, luminescence again decreasing between pH 4 and 2. Most interestingly, luminescence intensity and decay time simultaneously change with pH. Thus the design of luminescence decay time-based optical sensors for pH and gases such as carbon dioxide, which changes the pH of suitable buffer solutions, becomes possible. This is of special interest, since the europium complexes do not show a sensitivity of their luminescence to oxygen, which is in contrast to ruthenium dyes commonly used in luminescence decay time-based optical pH or CO2 sensors.